Timothy Petrie | University of Washington (original) (raw)
Papers by Timothy Petrie
Biological Interactions on Materials Surfaces, 2009
Page 1. 7 Extracellular Matrix-Derived Ligands for Selective Integrin Binding to Control Cell Fun... more Page 1. 7 Extracellular Matrix-Derived Ligands for Selective Integrin Binding to Control Cell Function Timothy A. Petrie and Andrés J. García Integrins are key cell surface receptors that function as the primary bridge between ...
Nanotechnology, 2012
Gold nanoparticles exhibiting surface plasmon resonances have been considered as photothermal age... more Gold nanoparticles exhibiting surface plasmon resonances have been considered as photothermal agents for the selective destruction of bacteria by visible to near-infrared radiation. Here, we consider theoretically the possible complementary use of sub-micron silicon carbide (SiC) particles as photothermal agents for the heating of bacteria by pulsed mid-infrared (MIR) radiation. A SiC microparticle can exhibit surface phonon resonances in the MIR. Similar to the effect of surface plasmon resonances in gold nanoparticles, this could lead to enhanced absorption at the resonant wavelength and strong heating of the microparticle locally. If the heating is sufficient, this might lead to damage of bacterial cells adjacent to SiC particles. We estimate the heating of sub-micron SiC particles in a water medium under various pulse lengths of radiation at wavelength 10.6 μm. Noting that SiC is being investigated as a biocompatible material that could be functionalized for biomedical applications, and that an appropriately roughened SiC surface could be expected to exhibit similar surface phonon resonances, we speculate that enhanced heating under MIR radiation may be useful for in vitro sterilization of such surfaces.
Journal of Bone and Mineral Research, 2014
Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogene... more Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (e.g., development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study we developed a model of BTx-induced muscle paralysis in adult zebrafish, and examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed latestage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra-and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders.
Biomacromolecules, 2013
Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide 2 unlimited... more Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide 2 unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We 4 present a novel approach to effectively enhance myogenic differentiation of human embryonic 5 stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble 6 the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a 7 protein. The myogenic differentiation was assessed by cell morphology, gene activities, and 8 protein expression. hESCs grown on C-PCL uniaxially-aligned nanofibers in media containing 9 Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber 10 orientation, with increased expressions of marker genes and proteins associated with myogenic 11 differentiation as compared to control substrates. The combination of Wnt3a signaling and 12 aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells 13 over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of 14 cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of 15 hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that 16 chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system 17 for embryonic myogenesis and muscle regeneration.
I lllll llllllll Il l lllll lllll lllll lllll lllll 111111111111111111111111111111111 c12) United... more I lllll llllllll Il l lllll lllll lllll lllll lllll 111111111111111111111111111111111 c12) United States Patent Garcia et al.
![Research paper thumbnail of Controlling Cell Adhesion to Titanium: Functionalization of Poly[oligo(ethylene glycol)methacrylate] Brushes with Cell-Adhesive Peptides](https://attachments.academia-assets.com/99448907/thumbnails/1.jpg)
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Science Translational Medicine, 2010
Titanium implants coated with nanoclustered ligands for integrin adhesion receptors are tightly i... more Titanium implants coated with nanoclustered ligands for integrin adhesion receptors are tightly integrated into bone for orthopedic applications.
Biomaterials, 2006
Polymethylmethacrylate (PMMA) bone cement serves as the primary fixation material between bone an... more Polymethylmethacrylate (PMMA) bone cement serves as the primary fixation material between bone and the prosthetic component in cemented total hip arthroplasty. In vivo degradation of bone cement may lead to a decrease in mechanical properties of PMMA and result in aseptic loosening. However, other factors such as porosity and location of the cement relative to the bone implant interface may also contribute to mechanical behavior in vivo. This study investigated the mechanical properties of Simplex s cement retrieved from 43 patients undergoing revision total hip arthroplasty. The time in vivo was between 1 month and 27 years. The variables studied included fracture toughness (K IC), porosity, molecular weight, time in vivo of the cement, and relative in vivo location of the cement with respect to the implant and bone. K IC did not correlate with time in vivo of the samples or with molecular weight. This suggests that time in vivo may not be the limiting factor in the mechanical integrity of the bone cement, A significant and inverse relationship was found between porosity and K IC. This implies that porosity is the most important factor in the mechanical behavior of bone cement during in vivo use.
Development (Cambridge, England), 2014
Neutrophils and macrophages, as key mediators of inflammation, have defined functionally importan... more Neutrophils and macrophages, as key mediators of inflammation, have defined functionally important roles in mammalian tissue repair. Although recent evidence suggests that similar cells exist in zebrafish and also migrate to sites of injury in larvae, whether these cells are functionally important for wound healing or regeneration in adult zebrafish is unknown. To begin to address these questions, we first tracked neutrophils (lyzC(+), mpo(+)) and macrophages (mpeg1(+)) in adult zebrafish following amputation of the tail fin, and detailed a migratory timecourse that revealed conserved elements of the inflammatory cell response with mammals. Next, we used transgenic zebrafish in which we could selectively ablate macrophages, which allowed us to investigate whether macrophages were required for tail fin regeneration. We identified stage-dependent functional roles of macrophages in mediating fin tissue outgrowth and bony ray patterning, in part through modulating levels of blastema pro...
Development (Cambridge, England), 2014
Neutrophils and macrophages, as key mediators of inflammation, have defined functionally importan... more Neutrophils and macrophages, as key mediators of inflammation, have defined functionally important roles in mammalian tissue repair. Although recent evidence suggests that similar cells exist in zebrafish and also migrate to sites of injury in larvae, whether these cells are functionally important for wound healing or regeneration in adult zebrafish is unknown. To begin to address these questions, we first tracked neutrophils (lyzC(+), mpo(+)) and macrophages (mpeg1(+)) in adult zebrafish following amputation of the tail fin, and detailed a migratory timecourse that revealed conserved elements of the inflammatory cell response with mammals. Next, we used transgenic zebrafish in which we could selectively ablate macrophages, which allowed us to investigate whether macrophages were required for tail fin regeneration. We identified stage-dependent functional roles of macrophages in mediating fin tissue outgrowth and bony ray patterning, in part through modulating levels of blastema pro...
Biointerphases, 2009
This review focuses on the surface modification of substrates with self-assembled monolayers ͑SAM... more This review focuses on the surface modification of substrates with self-assembled monolayers ͑SAMs͒ and polymer brushes to tailor interactions with biological systems and to thereby enhance their performance in bioapplications. Surface modification of biomedical implants promotes improved biocompatibility and enhanced implant integration with the host. While SAMs of alkanethiols on gold substrates successfully prevent nonspecific protein adsorption in vitro and can further be modified to tether ligands to control in vitro cell adhesion, extracellular matrix assembly, and cellular differentiation, this model system suffers from lack of stability in vivo. To overcome this limitation, highly tuned polymer brushes have been used as more robust coatings on a greater variety of biologically relevant substrates, including titanium, the current orthopedic clinical standard. In order to improve implant-bone integration, the authors modified titanium implants with a robust SAM on which surface-initiated atom transfer radical polymerization was performed, yielding oligo͑ethylene glycol͒ methacrylate brushes. These brushes afforded the ability to tether bioactive ligands, which effectively promoted bone cell differentiation in vitro and supported significantly better in vivo functional implant integration.
Journal of Cellular Physiology, 2008
Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is... more Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling. J. Cell. Physiol. 217: 450–458, 2008. © 2008 Wiley-Liss, Inc.
Biomacromolecules, 2009
Attaining control over the surface chemistry of titanium is critical to its use in medical implan... more Attaining control over the surface chemistry of titanium is critical to its use in medical implants, especially to address complications such as infection and loosening of implants over time, which still present significant challenges. The surface-initiated atom transfer radical polymerization (SI-ATRP) of a saccharide-substituted methacrylate, 2-gluconamidoethyl methacrylate (GAMA), affords dense polymer brushes that resist protein adsorption and cell adhesion. We further tailored the nature of the surfaces by covalent attachment of an adhesion peptide to afford control over cell adhesion. Whereas unmodified poly(GAMA) brushes prevent cell adhesion, brushes with a tethered GFOGER-containing peptide sequence promote the deposition of confluent well-spread cells. The presentation of adhesion proteins on a robust bioresistive background in this fashion constitutes a versatile approach to the development of new biomaterials.
Colloids and Surfaces B-biointerfaces, 2010
Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that re... more Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some particular chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network on the material surface. We have investigated the organization of a recombinant fragment of fibronectin (FNIII 7-10 ) upon adsorption on this particular chemistry, PEA. Atomic force microscopy (AFM) was used to identify individual molecules of the fragment after adsorption, as well as the evolution of the distribution of adsorbed molecules on the surface of the material as the concentration of the adsorbing solution increased. Globular molecules that turn into small aggregates were found as a function of solution concentration. Above a threshold concentration of the adsorbing solution (50 g/mL) an interconnected network of the FNIII 7-10 fragment is obtained on the material surface. The bioavailability of specific cell adhesion domains, including RGD, within the molecules was higher on PEA than on the control glass. The biological activity of the fragment was further investigated by evaluating focal adhesion formation and actin cytoskeleton for MC3T3-E1 osteoblast-like cells. Well-developed focal adhesion complexes and insertions of actin stress fibers were found on PEA in a similar way as it happens in the control SAM-OH. Moreover, increasing the hydrophilicity of the surface by incorporating -OH groups led to globular molecules of the fragment homogeneously distributed throughout the surface; and the cell-material interaction is reduced as depicted by the lack of well-developed focal plaques and actin cytoskeleton. (A.J. García), masalsan@fis.upv.es (M. Salmerón-Sánchez).
Biomacromolecules, 2008
A polylactide copolymer with pendant benzyloxy groups has been synthesized by the copolymerizatio... more A polylactide copolymer with pendant benzyloxy groups has been synthesized by the copolymerization of a benzyl-ether substituted monomer with lactide. Debenzylation of the polymer to provide pendant hydroxyl groups followed by modification with succinic anhydride affords the corresponding carboxylic acid functionalized copolymer that is amenable to standard carbodiimide coupling conditions to attach amine-containing biological molecules. An amino-substituted biotin derivative was coupled to the carboxyl functional groups of copolymer films as proof-of-concept. In a demonstration of the function of these new materials, an RGD-containing peptide sequence was tethered to copolymer films at various densities and was shown to enhance the adhesion of epithelial cells. This strategy provides the opportunity for the attachment of a variety of ligands, allowing for the fabrication of a versatile class of biodegradable, biocompatible materials.
Journal of Cellular and Molecular Medicine, 2009
Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device... more Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of FN-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed arginine–glycine–aspartic acid peptides, serum proteins and full-length FN. Differences in biological responses correlated with integrin binding specificity and signalling among surface coatings. This work validates a simple, clinically translatable, surface biofunctionalization strategy to enhance biomedical device integration.
Acta Biomaterialia, 2010
Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative ... more Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression and function during hMSC commitment along osteogenic, chondrogenic and adipogenic lineages. Using self-assembled monolayers of omega-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long-term functional differentiation of adult stem cells, and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.
Journal of Biomedical Materials Research Part A, 2009
Microcontact printing (μ-CP) is a facile, cost-effective, and versatile soft-lithography techniqu... more Microcontact printing (μ-CP) is a facile, cost-effective, and versatile soft-lithography technique to create two-dimensional patterns of domains with distinct functionalities that provides a robust platform to generate micropatterned biotechnological arrays and cell culture substrates. Current μ-CP approaches rely on nonspecific immobilization of biological ligands, either by direct printing or adsorption from solution, onto micropatterned domains surrounded by a nonfouling background. This technique is limited by insufficient control over ligand density. We present a modified μ-CP protocol involving stamping mixed ratios of carboxyl- and tri(ethylene glycol)-terminated alkanethiols that provides for precise covalent tethering of single or multiple ligands to prescribed micropatterns via standard peptide chemistry. Processing parameters were optimized to identify conditions that control relevant endpoint pattern characteristics. This technique provides a facile method to generate micropatterned arrays with tailorable and controlled presentation of biological ligands for biotechnological applications and analyses of cell–material interactions. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009
Biological Interactions on Materials Surfaces, 2009
Page 1. 7 Extracellular Matrix-Derived Ligands for Selective Integrin Binding to Control Cell Fun... more Page 1. 7 Extracellular Matrix-Derived Ligands for Selective Integrin Binding to Control Cell Function Timothy A. Petrie and Andrés J. García Integrins are key cell surface receptors that function as the primary bridge between ...
Nanotechnology, 2012
Gold nanoparticles exhibiting surface plasmon resonances have been considered as photothermal age... more Gold nanoparticles exhibiting surface plasmon resonances have been considered as photothermal agents for the selective destruction of bacteria by visible to near-infrared radiation. Here, we consider theoretically the possible complementary use of sub-micron silicon carbide (SiC) particles as photothermal agents for the heating of bacteria by pulsed mid-infrared (MIR) radiation. A SiC microparticle can exhibit surface phonon resonances in the MIR. Similar to the effect of surface plasmon resonances in gold nanoparticles, this could lead to enhanced absorption at the resonant wavelength and strong heating of the microparticle locally. If the heating is sufficient, this might lead to damage of bacterial cells adjacent to SiC particles. We estimate the heating of sub-micron SiC particles in a water medium under various pulse lengths of radiation at wavelength 10.6 μm. Noting that SiC is being investigated as a biocompatible material that could be functionalized for biomedical applications, and that an appropriately roughened SiC surface could be expected to exhibit similar surface phonon resonances, we speculate that enhanced heating under MIR radiation may be useful for in vitro sterilization of such surfaces.
Journal of Bone and Mineral Research, 2014
Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogene... more Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (e.g., development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study we developed a model of BTx-induced muscle paralysis in adult zebrafish, and examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed latestage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra-and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders.
Biomacromolecules, 2013
Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide 2 unlimited... more Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide 2 unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We 4 present a novel approach to effectively enhance myogenic differentiation of human embryonic 5 stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble 6 the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a 7 protein. The myogenic differentiation was assessed by cell morphology, gene activities, and 8 protein expression. hESCs grown on C-PCL uniaxially-aligned nanofibers in media containing 9 Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber 10 orientation, with increased expressions of marker genes and proteins associated with myogenic 11 differentiation as compared to control substrates. The combination of Wnt3a signaling and 12 aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells 13 over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of 14 cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of 15 hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that 16 chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system 17 for embryonic myogenesis and muscle regeneration.
I lllll llllllll Il l lllll lllll lllll lllll lllll 111111111111111111111111111111111 c12) United... more I lllll llllllll Il l lllll lllll lllll lllll lllll 111111111111111111111111111111111 c12) United States Patent Garcia et al.
Science Translational Medicine, 2010
Titanium implants coated with nanoclustered ligands for integrin adhesion receptors are tightly i... more Titanium implants coated with nanoclustered ligands for integrin adhesion receptors are tightly integrated into bone for orthopedic applications.
Biomaterials, 2006
Polymethylmethacrylate (PMMA) bone cement serves as the primary fixation material between bone an... more Polymethylmethacrylate (PMMA) bone cement serves as the primary fixation material between bone and the prosthetic component in cemented total hip arthroplasty. In vivo degradation of bone cement may lead to a decrease in mechanical properties of PMMA and result in aseptic loosening. However, other factors such as porosity and location of the cement relative to the bone implant interface may also contribute to mechanical behavior in vivo. This study investigated the mechanical properties of Simplex s cement retrieved from 43 patients undergoing revision total hip arthroplasty. The time in vivo was between 1 month and 27 years. The variables studied included fracture toughness (K IC), porosity, molecular weight, time in vivo of the cement, and relative in vivo location of the cement with respect to the implant and bone. K IC did not correlate with time in vivo of the samples or with molecular weight. This suggests that time in vivo may not be the limiting factor in the mechanical integrity of the bone cement, A significant and inverse relationship was found between porosity and K IC. This implies that porosity is the most important factor in the mechanical behavior of bone cement during in vivo use.
Development (Cambridge, England), 2014
Neutrophils and macrophages, as key mediators of inflammation, have defined functionally importan... more Neutrophils and macrophages, as key mediators of inflammation, have defined functionally important roles in mammalian tissue repair. Although recent evidence suggests that similar cells exist in zebrafish and also migrate to sites of injury in larvae, whether these cells are functionally important for wound healing or regeneration in adult zebrafish is unknown. To begin to address these questions, we first tracked neutrophils (lyzC(+), mpo(+)) and macrophages (mpeg1(+)) in adult zebrafish following amputation of the tail fin, and detailed a migratory timecourse that revealed conserved elements of the inflammatory cell response with mammals. Next, we used transgenic zebrafish in which we could selectively ablate macrophages, which allowed us to investigate whether macrophages were required for tail fin regeneration. We identified stage-dependent functional roles of macrophages in mediating fin tissue outgrowth and bony ray patterning, in part through modulating levels of blastema pro...
Development (Cambridge, England), 2014
Neutrophils and macrophages, as key mediators of inflammation, have defined functionally importan... more Neutrophils and macrophages, as key mediators of inflammation, have defined functionally important roles in mammalian tissue repair. Although recent evidence suggests that similar cells exist in zebrafish and also migrate to sites of injury in larvae, whether these cells are functionally important for wound healing or regeneration in adult zebrafish is unknown. To begin to address these questions, we first tracked neutrophils (lyzC(+), mpo(+)) and macrophages (mpeg1(+)) in adult zebrafish following amputation of the tail fin, and detailed a migratory timecourse that revealed conserved elements of the inflammatory cell response with mammals. Next, we used transgenic zebrafish in which we could selectively ablate macrophages, which allowed us to investigate whether macrophages were required for tail fin regeneration. We identified stage-dependent functional roles of macrophages in mediating fin tissue outgrowth and bony ray patterning, in part through modulating levels of blastema pro...
Biointerphases, 2009
This review focuses on the surface modification of substrates with self-assembled monolayers ͑SAM... more This review focuses on the surface modification of substrates with self-assembled monolayers ͑SAMs͒ and polymer brushes to tailor interactions with biological systems and to thereby enhance their performance in bioapplications. Surface modification of biomedical implants promotes improved biocompatibility and enhanced implant integration with the host. While SAMs of alkanethiols on gold substrates successfully prevent nonspecific protein adsorption in vitro and can further be modified to tether ligands to control in vitro cell adhesion, extracellular matrix assembly, and cellular differentiation, this model system suffers from lack of stability in vivo. To overcome this limitation, highly tuned polymer brushes have been used as more robust coatings on a greater variety of biologically relevant substrates, including titanium, the current orthopedic clinical standard. In order to improve implant-bone integration, the authors modified titanium implants with a robust SAM on which surface-initiated atom transfer radical polymerization was performed, yielding oligo͑ethylene glycol͒ methacrylate brushes. These brushes afforded the ability to tether bioactive ligands, which effectively promoted bone cell differentiation in vitro and supported significantly better in vivo functional implant integration.
Journal of Cellular Physiology, 2008
Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is... more Cell adhesion to extracellular matrix (ECM) components through cell-surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non-adhesive surfaces presenting well-defined, independent densities of two integrin-specific engineered ligands for the type I collagen (COL-I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross-talk on adhesive responses. Engineered surfaces displayed ligand density-dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL-I ligand surfaces. Moreover, surfaces presenting mixed COL-I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling. J. Cell. Physiol. 217: 450–458, 2008. © 2008 Wiley-Liss, Inc.
Biomacromolecules, 2009
Attaining control over the surface chemistry of titanium is critical to its use in medical implan... more Attaining control over the surface chemistry of titanium is critical to its use in medical implants, especially to address complications such as infection and loosening of implants over time, which still present significant challenges. The surface-initiated atom transfer radical polymerization (SI-ATRP) of a saccharide-substituted methacrylate, 2-gluconamidoethyl methacrylate (GAMA), affords dense polymer brushes that resist protein adsorption and cell adhesion. We further tailored the nature of the surfaces by covalent attachment of an adhesion peptide to afford control over cell adhesion. Whereas unmodified poly(GAMA) brushes prevent cell adhesion, brushes with a tethered GFOGER-containing peptide sequence promote the deposition of confluent well-spread cells. The presentation of adhesion proteins on a robust bioresistive background in this fashion constitutes a versatile approach to the development of new biomaterials.
Colloids and Surfaces B-biointerfaces, 2010
Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that re... more Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some particular chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network on the material surface. We have investigated the organization of a recombinant fragment of fibronectin (FNIII 7-10 ) upon adsorption on this particular chemistry, PEA. Atomic force microscopy (AFM) was used to identify individual molecules of the fragment after adsorption, as well as the evolution of the distribution of adsorbed molecules on the surface of the material as the concentration of the adsorbing solution increased. Globular molecules that turn into small aggregates were found as a function of solution concentration. Above a threshold concentration of the adsorbing solution (50 g/mL) an interconnected network of the FNIII 7-10 fragment is obtained on the material surface. The bioavailability of specific cell adhesion domains, including RGD, within the molecules was higher on PEA than on the control glass. The biological activity of the fragment was further investigated by evaluating focal adhesion formation and actin cytoskeleton for MC3T3-E1 osteoblast-like cells. Well-developed focal adhesion complexes and insertions of actin stress fibers were found on PEA in a similar way as it happens in the control SAM-OH. Moreover, increasing the hydrophilicity of the surface by incorporating -OH groups led to globular molecules of the fragment homogeneously distributed throughout the surface; and the cell-material interaction is reduced as depicted by the lack of well-developed focal plaques and actin cytoskeleton. (A.J. García), masalsan@fis.upv.es (M. Salmerón-Sánchez).
Biomacromolecules, 2008
A polylactide copolymer with pendant benzyloxy groups has been synthesized by the copolymerizatio... more A polylactide copolymer with pendant benzyloxy groups has been synthesized by the copolymerization of a benzyl-ether substituted monomer with lactide. Debenzylation of the polymer to provide pendant hydroxyl groups followed by modification with succinic anhydride affords the corresponding carboxylic acid functionalized copolymer that is amenable to standard carbodiimide coupling conditions to attach amine-containing biological molecules. An amino-substituted biotin derivative was coupled to the carboxyl functional groups of copolymer films as proof-of-concept. In a demonstration of the function of these new materials, an RGD-containing peptide sequence was tethered to copolymer films at various densities and was shown to enhance the adhesion of epithelial cells. This strategy provides the opportunity for the attachment of a variety of ligands, allowing for the fabrication of a versatile class of biodegradable, biocompatible materials.
Journal of Cellular and Molecular Medicine, 2009
Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device... more Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of FN-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed arginine–glycine–aspartic acid peptides, serum proteins and full-length FN. Differences in biological responses correlated with integrin binding specificity and signalling among surface coatings. This work validates a simple, clinically translatable, surface biofunctionalization strategy to enhance biomedical device integration.
Acta Biomaterialia, 2010
Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative ... more Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression and function during hMSC commitment along osteogenic, chondrogenic and adipogenic lineages. Using self-assembled monolayers of omega-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long-term functional differentiation of adult stem cells, and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.
Journal of Biomedical Materials Research Part A, 2009
Microcontact printing (μ-CP) is a facile, cost-effective, and versatile soft-lithography techniqu... more Microcontact printing (μ-CP) is a facile, cost-effective, and versatile soft-lithography technique to create two-dimensional patterns of domains with distinct functionalities that provides a robust platform to generate micropatterned biotechnological arrays and cell culture substrates. Current μ-CP approaches rely on nonspecific immobilization of biological ligands, either by direct printing or adsorption from solution, onto micropatterned domains surrounded by a nonfouling background. This technique is limited by insufficient control over ligand density. We present a modified μ-CP protocol involving stamping mixed ratios of carboxyl- and tri(ethylene glycol)-terminated alkanethiols that provides for precise covalent tethering of single or multiple ligands to prescribed micropatterns via standard peptide chemistry. Processing parameters were optimized to identify conditions that control relevant endpoint pattern characteristics. This technique provides a facile method to generate micropatterned arrays with tailorable and controlled presentation of biological ligands for biotechnological applications and analyses of cell–material interactions. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009